Diagnostic composition and method



Patented July 21, 1942 DIAGNOSTIC COMPOSITION AND .METHOD I JonasKamiet, Brooklyn, N. Y., assignor to Miles Laboratories, Inc., Eikhart,Ind., a corporation of Indiana No Drawing.

'lClaims.

The present invention relates to diagnostic compositions and, moreparticularly, to diagnostic compositions suitable for use in thequalitative detection and semi-quantitative estimation of blood in bodyfluids, such as urine, vomitus, gastric contents, semen, cerebrospinalfluid and in feces. It has for its object to provide a simple, rapid andconvenient method for performing this test with a high degree ofaccuracy and specificity, and one that can readily be used by theaverage physician without laboratory equipment, supplies of freshchemicals or specialized analytical training.

The detection of occult blood in body fluids and feces has become aninvaluable aid to the medical practitioner in the correct diagnosis of agreat number of disorders. Blood is found in the gastric contents and invomitus in conditions associated with erosion of the mucou membranes, inulcers and in carcinomas. regular and frequent occurrence of occultblood is suggestive of gastro intestinal cancer, gastric or duodenalulcers or hemorrhoids. In these conditions, the hemorrhage is often soslight that it is not possible to detect blood by microscopicidentifications of the erythrocytes (red blood cells) and a sensitiveand specific chemical test for occult blood becomes invaluable. In theurine, blood cells (hematuria) or blood pigment hemoglobinuria) is foundin typhus, scurvy, purpura, pyemia, nephritis, renal calculi, as there-,- sult of a burn covering a large part of the body, by the action ofvarious hemolytic toxins, etc.

However, the methods heretofore employed for the detection of occultblood require some technical ability, experience and fresh supplies ofreagents which are not always available to the average physician. Thus,the benzidine reaction which is capable of great accuracy and is one ofthe most delicate tests for the detection of occult blood, requires thata fresh solution of benzidine in glacial acetic acid be prepared daily.Since these solutions change rapidly, especially in contact with light,they rapidly lose their sensitivity and must be discarded. (Journal ofBiological Chemistry, volume 19, page 445, 1914; Zentralblatt furChirurgie, volume 41, No. 28, 1914.)

Ruttan and Hardisty (Canadian Medical Association Journal, Nov. 1912;Biochemical Bulletin, volume 2, page 225, 1913) have described amodification of the benzidine reaction, using o-tolidine dissolved inglacial acetic acid. This solution is said to remain stable for onemonth. It has, however, been my experience that after one week, thesolution of o-tolidine becomes too In the feces, the

Application September 26, 1940, Serial No. 358,480

insensitive for use since it will no longer detect traces of occultblood that will give a definite positive reaction with a fresho-tolidine solution.

The benzidine reaction for the detection of occult blood is based, ingeneral, on the reaction of the peroxidase present in the blood with thehydrogen peroxide or the reagent to form active" oxygen. The benzidineacts as an oxygen acceptor and is readily oxidized to a coloredquinonoid form. In general, any compound of the 7 general formula:

mN o r Ni where A, B, C, D, E, F, G and H are members of the groupconsisting of hydrogen and alkyl radicals, may be used as an oxygenacceptor in the benzidine reaction. Examples of such members arebenzidine, orthotolidine, metatoli dine, etc.

The present invention has for its further ob ject to provide a meanswhereby an unskilled tion which remains stable and sensitive over longperiod of time under all ordinary conditions of temperature andhumidity. The basis of the present invention is the finding that suchcompositions of high sensitivity, good specificity and great stabilitycan be obtained by mixing, in the dry state:

(a). A compound of the general formula:

HzN NP:

where A, B, C, D, E, F, G and H are members of the group consisting ofhydrogen and alkyl radicals.

(b) A member a of the group comprising the peroxides, perborates andpersulfates ofthe alwithout the use of i .sodium bisulfate, etc., etc.Members 01' this group, when reacted in aqueous solution with a memberof the group consisting of the peroxides,

perborates and persulfates of the alkali metals and the alkali-earthmetals, will form hydrogen peroxide at a slow and steady rate.

To determine qualitatively whether occult blood is present in aspecimen, one drop of the material to be tested (in the case of feces-anaqueous suspension of the specimen) is deposited on a measured quantityof thedry composition, e. g. a five-grain tablet. In the presence ofblood, a blue or green colored spot will form within a short time. If,after five minutes, there is no change of color, the specimen is free01' occult blood. Alternatively, the dry composition may be suspended inwater and mixed with the material to be tested. In the presence ofoccult blood, a blue or green color will be obtained within fiveminutes.

A semi-quantitative estimation of the amount of occult blood present inthe specimen may be made by measuring the time required for the firstdefinite blue or green coloration to be observable. A definitecoloration within thirty seconds is reported as four plus, thirtyseconds to one minutethree plus, one minute to two minutestwo plus, twominutes to three minutes-one plus,

three minutes to five minutes-plus minus. If

no definite coloration is obtained after five minutes, the test isreported as negative.

' It is obvious, of course, that diluents, excipients, dispersingagents, auxiliary substances and coating materials may be incorporatedinto these compositions without changing the basis of the presentinvention. Diluents such as talc, kaolin and gypsum may be added toobtain a tablet with better keeping qualities, or a more stable product.Excipients such as lactose, starch or flour may be added to facilitatethe formation of granules. Dispersing agents such as saponin, gelatin,agar, soap, alkali-metal silicates, tragacanth or gum arabic may beadded to accelerate the disintegration of the tablet when placed intofluid. Auxiliary agents, such as coloring matters or perfumes maylikewise be added. To improve the keeping qualitiesof these compositionsin tablet form, they may be covered with a water-dispersible varnish orwax. These compositions may be used in the form or powder, or granules,or dispensed in capsules, or they may be pressed into tablets of suchsize is convenient for the peri'ormance 01! a sin le te The followingexamples of typical compositions covered by this invention are intendedto define and illustrate but in no way to limit this invention to thereagents, proportions or conditions described therein. Obviousmodifications will occur to any *person skilled in the art.

Example I Grams Anhydrous granular citric acid 1000 Benzidine,chemically pure 100 Barium peroxide, heavy powder 250 Talc powder 250are intimately mixed until homogeneous. The mixture is then compressedinto five-grain tablets.

The sensitivity of this composition is such that it will detect one partof blood in one million parts of urine or one and a half million partsof water. Example II Grams Sodium acid pyrophosphate 1000 Orthotolidine,chemically pure 50 Sodium perborate monohydrate 200 Kaolin powder 500are intimately mixed until homogeneous. The

mixture is then compressed into five-grain tablets.

The sensitivity of this composition is such that it will detect one partof blood in eight hundred thousand parts of urine or one million partsof water.

Having described my invention, what I claim and desire to protect byLetters Patent is:

1. Diagnostic compositions comprising in dry solid form a member of thegroup consisting oi benzidine, ortho-tolidine and metatolidine, a memberof the group consisting of the peroxides, perborates and persulfates ofthe alka i metals and the alkali-earth metals and a solid compound whichcontains at least one available acidic hydrogen atom.

2. Diagnostic compositions comprising in dry solid form a member of thegroup consisting of benzidine, ortho-tolidine and meta-tolidine, bariumperoxide and a solid compound which contains at least one availableacidic hydrogen atom.

3. Diagnostic compositions comprising in dry solid form a member of thegroup consisting of benzidine, ortho-tolidine and meta-tolidine, bariumperoxide and citric acid.

4. Diagnostic compositions comprising in dry solid form benzidine,barium peroxide and citric acid.

5. Diagonstic compositions comprising in dry solid form orth-tolidine,sodium perborate and sodium acid pyrophosphate.

6. A process for testing for blood in fluids which consists in adding ameasured amount or said fiuid to a measured amount of a compositioncomprising in dry solid form a member of the group consisting ofbenzidine, ortho-tolidine and meta-tolidine, a member of the groupconsisting of the peroxides, perborates and persulfates of the alkalimetals and the alkali-earth metals, and a solid compound which containsat least one available acidic hydrogen atom, whereby a detectablecolor-change results.

7. A process for testing for blood in fluids which consists in mixing ameasured amount of said fluid with a suspension in water of a measuredamount of a composition comprising in dry solid form a member of thegroup consisting of benzidine, ortho-tolidine and meta-tolidine, amember of the group consisting of the peroxides, perborates andpersuliates of the alkali metals and the alkali-earth metals, and asolid compound which contains at least one available acidic hydrogenatom, whereby a detectable color-change results.

JONAS KAMLET.

